1. Field of the Invention
The present invention relates to a modified glutamine:fructose-6-phosphate amidotransferase, which can be rapidly purified in quantities sufficient for the screening of compounds modifying its activity.
2. Description of the Related Art
Glutamine:fructose-6-phosphate amidotransferases (GFAT), EC 2.6.1.16, also called glucosamine-6-phosphate synthases or 2-deoxy-glucose-6-phosphate ketol isomerases, are involved in the biosynthesis route of hexosamines. GFAT catalyzes the first, limiting, stage of this biosynthesis route according to the reaction:L-Glutamine+fructose-6-phosphate→L-Glutamate+glucosamine-6-phosphateby transfer of the amidic nitrogen from the L-Glutamine to the ketone function of the fructose-6-phosphate. The GFATs therefore control the flow of glucose in the route of the hexosamines, via the fructose-6-phosphate, and consequently the formation of the hexosamines produced.
A recombinant bacterial form of GFAT, the glucosamine-6-phosphate synthase of Escherichia coli, has been purified to homogeneity and studied exhaustively. The properties and the enzymatic mechanism of the amide transfer have in particular been widely described (article by Teplyakov et al., Nat. Prod. Rep. (2002) 19:60). In particular, this enzyme, the crystalline structure of which has been resolved (Teplyakov et al., J. Mol. Biol. (2001) 313:1093), is formed by two domains, one having a hydrolase activity (glutaminase) and the other an isomerase activity.
Moreover, eukaryotic GFATs have been characterized, including in particular that of rat liver (Huynh et al., Arch. Biochem. Biophys. (2000) 379:307) and that of the yeast Candida albicans (Milewsky et al., J. Biol. Chem. (1999) 274:4000).
In humans, preliminary studies have shown the presence of GFAT activity in the liver (Ghosh et al., J. Biol. Chem. (1960) 235:1265). Several GFATs are now known. GFAT1, the principal form, GFAT2, which is preferentially expressed in the central nervous system, and GFAT1Alt, an isoform of GFAT1, essentially expressed in the striated muscles. The peptide sequences of GFAT1 and GFAT2 possess 75% sequence identity with each other, and those of GFAT1 and GFAT1Alt are identical except for an insertion of 18 amino acids into the GFAT1Alt sequence. The sequences of GFAT are therefore very preserved in humans, but also between species, since the peptide sequences of human GFAT1 and E. coli GFAT or mouse GFAT1 have 35% and 99% identity respectively.
The human GFAT1 gene was cloned in 1992 (McKnight et al., J. Biol. Chem. (1992) 267:25208). It codes a protein of 77 kDa formed by two distinct domains (Teplyakov et al., Nat. Prod. Rep. (2002) 19:60).
The increase in the production of UDP-NAc-GlcNH2, the final product of the biosynthesis route of the hexosamines, and its accumulation in the tissues have recently been involved in the development of insulin-resistance (Marshall et al., FASEB J. (1991) 5:3031, Yki-Jarvinen et al., Diabetes (1996) 45:302, Thompson et al., J. Biol. Chem. (1997) 272: 7759, Hawkins et al., J. Clin. Invest. (1997) 99:2173, Robinson et al., Diabetes (1993) 42:1333, Daniels et al., J. Clin. Invest. (1995) 96:1235, Baron et al., J. Clin. Invest. (1995) 96:2792).
Thus, it has been shown that an increase in the cell level of UDP-NAc-GlcNH2 by a slight overexpression of GFAT1, or a supply of exogenic glucosamine, can induce insulin-resistance both in vivo and in adipocytes in culture (Robinson et al., Diabetes (1993) 42:1333, Daniels et al., J. Clin. Invest. (1995) 96:1235, Baron et al., J. Clin. Invest. (1995) 96:2792, Hebert et al., J. Clin. Invest. (1996) 98:930).
In fact, insulin activates its transduction route by binding to its receptor, which induces the translocation of the glucose transporters, such as the GLUT4 receptor, stored in the cell, towards the membrane, and increases the inflow of glucose. The glucose thus enters the glycolysis route and is converted to glucose-6-phosphate then to fructose-6-phosphate. When the inflow of glucose is excessive, the fructose-6-phosphate enters the biosynthesis route of the hexosamines and is converted to glucosamine-6-phosphate by the GFAT. Several observations indicate that the metabolites of the glucosamine-6-phosphate prevent the translocation of the glucose receptors towards the cell membrane, thus reducing the inflow of the cell glucose (Marshall et al., FASEB J. (1991) 5:3031, Giacarri et al., Diabetologia (1995) 38:518, Marshall et al., J. Biol. Chem. (1991) 266:4706, Paterson et al., Endocrinology (1995) 136:2809).
The mechanism by which the metabolites of the glucosamine-6-phosphate exercise their physiological effects is not clear. One hypothesis has however been proposed: a high cytosolic concentration of UDP-NAc-GlcNH2 would lead to the hyperglycosylation of the Ser or Thr phosphorylation sites, thus leading to the stopping of the insulin-signalling route (Comer et al., J. Biol. Chem. (2000) 275:29179).
The GFAT activity is therefore considered as being one of the causes of high levels of blood glucose; moreover it is known to be high in patients suffering from non-insulin-dependant sugar diabetes or type II diabetes (Yki-Jarvinen et al., Diabetes (1996) 45:302).
Obtaining GFAT inhibitors would make it possible to reduce glycaemia in particular in individuals suffering from pathologies linked to hyperglycaemia, such as type II diabetes, acidosis and/or diabetic ketosis, for example.
Fungal or plant GFAT inhibitors could also make it possible to obtain fungicides and herbicides respectively.
However, in spite of the obtaining of recombinant forms of GFAT, the instability of the enzymatic preparations obtained, their small quantity, and their insufficient purification level, have not made it possible to obtain effective GFAT inhibitors.